Depletion of intracellular putrescine and spermidine by α-difluoromethylornithine does not inhibit proliferation of 9L rat brain tumor cells

Vol. 86, No. 4, 1979 February 28, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 1192-1198

DEPLETION OF INTRACELLULAR PUTRESCINE AND SPERMIDINE BY o-DIFLUOROMETHYLORNITHINE DOES NOT INHIBIT PROLIFERATION OF 9L RAT BRAIN TUMOR CELLS

Jerome Seidenfeld2and Laurence J. Marton Brain Tumor ResearchCenter Department of Neurological Surgery University of California School of Medicine San Francisco, CA 94143 Received January 16, 1979 Incubation of 9L rat brain tumor cells with 25 mM DL-adifluoromethylornithine inhibits cell proliferation, while treatment with 10mM and 1 mM do not. All three concentrations cause equal degreesof depletion of intracellular putrescine and spermidine content, but have no effect on sperminecontent. These observations showthat 9L cells can continue to proliferate in spite of significant polyamine depletion and leadsone to question the role of polyamines in 9L cell replication. Theseobservations also suggestthat inhibition of 9L cell proliferation by 25 mM DL- cr-difluormethylornithine is probably not due to its effect on ornithine decarboxylase or on intracellular polyamine content. INTRODUCTION The current interest in elucidating the intracellular role(s) of the polyamines has led to the synthesisand investigation of a number of inhibitors of L-ornithine decarboxylase (ODC4, E.C.4.1.1.17)(1-Ii),the first enzyme in the polyamine biosynthetic pathway (12,131.Metcalf, et al showed(9) that DL-cGdifluoromethylornithine (DFMO), first synthesized by Bey and Vevert (81, is an enzyme-activated irreversible inhibitor of mammalianODC. Mamont, et al report (7) that DFMO treatment results in inhibition of proliferation of rat hepatoma, L1210murine leukemia, 1 . This work was supported by NC1 grants CA-15515 and CA-13525 and a gift from the Phi Beta Psi Sorority. LJM is the recipient of NC1 Research Career Develgpment Award CA-00 112. 3To whom reprint requests shouldbe addressed. 4Also in the Department of Laboratory Medicine. The abbreviations used are: ODC, ornithine decarboxylase; DFMO, DLGdifluoromethylornithine; Pu, putrescine; Sd, spermidine; Sp, spermine; HBSS,Hank’s balanced salt solution; neq, nanoequivalents;aMO, DL-a-methylornithine. 0006-291

X/79/041

192-07$01

.OO/ 0

Copyright @I I979 by Academic Press, Inc. All rights of reproduction in any form reserved.

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BIOCHEMICAL

and human prostatic effect

adenoma

cells --in vitro.

of DFMO to the observed

spermidine

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

decline

These authors

in intracellular

(Sd). DFMO was also shown (11) to prolong

of mice incubated

with L1210 leukemic

observations

on the effects

MATERIALS

AND METHODS

of DFMO

cells.

attribute

putrescine

this cytostatic (Pu) and

the mean survival

We report

time

here some preliminary

on VL rat brain tumor

cells grown --in vitro.

All chemicals used were of analytical grade. The DFMO used in this study was the generous gift of Merrell International Research Center, Strasbourg, France. Rat 9L brain tumor cells were grown in vitro 2s previously descrped (14). Cells were seeded into plastic Falcon flasks775 cm surface area, 10 cells/flask) in 13.5 ml of medium (14) 24-26 hours before starting an experiment, to allow cells to reach exponential growth before treatment (15). Cells were treated with 1.5 ml of a 10 fold concentrated solution of DFMO in Hank’s balanced salt solution (HBSS), adjusted to pH 7.2, that when added to the flask was diluted to the appropriate concentration. Control flasks were treated with 1.5 ml of HBSS. At various times after treatment the incubation medium was decanted, the cegs were haryested by trypsinization (0.25% trypsin and 0.02% Na EDTA in Ca+ -and Mg+ -free HBSS for 5 min at 37 O) and suspended in 12 m f of Ice . cold 0.02% Na2 EDTA in HBSS. Three flasks treated with each concentration of DFMO and three control flasks were harvested at each time point. The total number of cells/flask was measured by counting an aliquot of each cell suspension using a Royce Cell-Crit model 927-TC counter (Royce Instruments, Menlo Park, CA). To measure the intracellular polyamine content 6 ml of each cell suspension was sedimented by centrifugatpn and the cell pellet was sonicated in 8% 5-sulfosalicylic acid (250 @/IO cells). The homogenate was kept on ice for one hour to precipitate protein, centrifuged (8,000 xg for 10 min at 4’) and 50 ~1 of supernatant was analyzed for polyamine content using a Durrum Instruments Inc. (Sunnyvale, CA) D-500 amino acid analyzer as previously described (16) with the following modifications. An 11.5 cm (1.75mm i.d.1 stainless steel column was packed with a 16% cross-linked 10: pm diameter sulfonated polystyrene cation exchange resin (Durrum Chemical Co., Sunnyvale, CA). Four buffers used for sequential elution of the polyamines were prepared as follows. A stock buffer (Buffer IV), 2.40N KC1 and O.OVN potassium citrate (pH 5.56), to which 12 ml/l thiodiglycol and 0.5 ml/l liquified phenol were added, was diluted to prepare Buffers I-III as follows (stock:water): I, 1:4; II, 1:l; III, 4:l. The three polyamines were quantitated by fluorescense measurement of the chromophore produced by reaction with o-phthal aldehyde. RESULTS The effects Figure

of 25, 10, and 1 mM DFMO on VL cell proliferation

1. At 48 and 72 hours post treatment

only 65% and 52% as many cells respectively concentrations

of DFMO

produced

flasks containing as did control

much smaller

1193

degrees

are shown in

25 mM DFMO had flasks.

of inhibition

The lower of cell

Vol. 86, No. 4, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

6-

4.0 B 2 u 2i 5

3.6

2.4

Figure 1: Effect of DFMO on 9L cell proliferation. Rat 9L brain tumor cells were incubated in the absence (0) or in the presence of 1.0 mM (01, 10.0 mM (m), or 25.0 mM (A) DFMO. Three flasks at each concentration were harvested by trypsinization at each time point and counted using a Royce Cell-Crit Model 927-TC cell counter. Points represent the mean (+ SD) of three determinations. Figure

2: Effect of DFMO on 9L intracellular putrescine content. Rat 9L brain incubated in the absence (01 or in the presence of 1.0 mM (01, , or 25.0 mM (A) DFMO. Three flasks at each concentration were at each time point. Cells were pelleted, sonicated in 8% 5-sulfosalicylic acid, spun at 8000 x g and aliquots of the supernatants were analyzed for intracellular putrescine content with a Durrum D-500 amino acid analyzer. Points represent the mean (+ SD) of three determinations.

proliferation.

By 96 hours flasks with 10 or 1 mM DFMO had as many cells as did

controls. Figure

2 shows that all three concentrations

Pu content

to less than 0.1 nmol/106

lower

of detection

limit

than 2% of control cells did not increase Treatment

and analytical

at the same time.

during the remainder

with DFMO also resulted

(Fig. 3) which was maximal treatment

cells by 12 hours after

of our preparative

cells’ Pu content

intracellular

treatment. methods.

This is the It is also less

The Pu content

of treated

of the 96 hour study. in depletion

(to less than 4% of control

for all three concentrations

was faster for cells treated

of DFMO depleted

studied.

of intracellular

Sd content

levels) at 48 hours post

The rate of decline

of Sd eontent

with 25 mM DFMO than for those incubated

with lower

concentrations. Figure spermine

4 shows that there were no statistically

(Sp) content

between

control

significant

cells and cells treated

1194

differences

in

with 25, 10, or 1 mM

Vol. 86, No. 4, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

4 2 3

3

“0 7 %

2

4-

s l-

1 -l

03

20

40 HOURS 60

80

100

III

04

1 20

11

I

40 HOURS 60

[ 80

Figure 3: Effect of DFMO on VL intrac&ular spermidine content. Rat VL brain tumor cells were incubated in the absence (0) or in the presence of 1.0 mM (@I, 10.0 mM (m, or 25.0 mM (A DFMO. Three flasks at each concentration were harvested by trypsinization at each time point. Cells were pelleted, sonicated in 8% 5-sulfosalicylic acid, spun at 8000 x g and aliquots of the suspernatants were analyzed for intracellular spermidine content with a Durrum D-500 amino acid analyzer. Points represent the mean (+ SD) of three determinations. Figure 4: Effect of DFMO on VL intracellular spermine content. Rat VL brain tumor cells were incubated in the absence (0) or in the presence of 1.0 mM (a), 10.0 mM (m, or 25.0 m.M (A)DFMO. Three flasks at each concentration were harvested by trypsinization at each time point. Cells were pelleted, sonicated in 8% 5-sulfosalicylic acid, spun at 8000 x g, and aliquots of the supernatant were analyzed for intracellular spermine content with a Durrum D-500 amino acid analyzer. Points represent the mean (+ SD) of three determinations.

DFMO.

The total polyamine

content

sum of Sd content

plus Sp content

above-mentioned

concentrations

(in nanoequivalents

(neq )/lo6

cells) and the

(in the same units), for cells treated of DFMO, are shown in Figures

with the

5 and 6 respectively.

DISCUSSION Within

48 hours of incubation

of VL cells with 10 or 1 mM DFMO the cells lost

more than 98% and 96% of their normal 3). Nevertheless,

the parallel

hours post treatment

growth

these depleted

curves obtained

this is the first report

proliferate

at the same rate as controls complement

respectively

(Figs 2 and

show that from 48 to 72

cells grew at the same rate as controls

We believe

its normal

Pu and Sd content

of a wild-type

cell line that continues

in spite of depletion

of two of the three polyamines.

1195

(Fig. 1).

to

of more than 95% of

In addition,

the lack of

1

t 100

Vol.

40

86,

No.

4, 1979

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

r

tl 20

40

60

100

80

HOURS

I

I

I

20

-

I

I

40

I

60

I 80

HOURS

w

Figure 5: Effect of DFMO on VL intracellular total polyamine content. Rat VL brain tumor cells were incubated in the absence (0) or in the presence of 1.0 mM (01, 10.0 mM (m or 25.0 mM (& DFMO. Data skwn in Figures 2, 3, and 4 were used to calculate total polyamine content in neq/lO cells as (2 x Pu content) + (3 x Sd content) + (4 x Sp content). Points represent the mean (+ SD) of three determinations. Figure 6: Effect of DFMO on VL intracellular Sd plus Sp content. Tumor cells were incubated in the absence (0) or in the presence of 1.0 mM (e), 10.0 mM (m, or 25.0 mM (A) DFMO. Data shgwn in Figures 3 and 4 were used to calculate total Sd plus Sp content in neq/lO cells as (3 x Sd content) + (4 x Sp content). Points represent the mean (2 SD) of three determinations.

correlation action

of the degree of polyamine

for the three concentrations

proliferation but rather

to some non-ODC

and polyamine

control

of DFMO studied

levels.

We hypothesized

content

on VL cells, However,

of 10 mM

for controls

oM0,

(aM0) which

did not have a

Sp content

to 150% of controls.

CrMO to inhibit

VL cell proliferation,

to replicate

might possibly

of neq/106 cells of Sd and Sp. This parameter and cells treated

cells of all three polyamines

of VL cells,

on VL cell proliferation

could be due to the compensatory

that the cell’s ability

by the total number

content

of

Pu and Sd to less than 5% and 33% of

eM0 increased

(17) that the failure

that inhibition

on polyamine

of o-methylornithine

depleted

10 mM

implies

action.

(17) showed that 10 mM

in spite of Pu and Sd depletion, Sp and proposed

specific

work on the effects

effect

not differ

with the degree of cytostatic

by DFMO may not be due to effects

Previous

cytostatic

depletion

did decline

with 10 mM for treated

1196

aM0 although cells.

increase

in

be determined did total neq/106

I

I 100

Vol. 86, No. 4, 1979

The results hypothesis. decrease

BIOCHEMICAL

we report

Figures

here, however,

both total

resulting

clearly

polyamine

content

Sd depletion

at the same rate as controls.

complement

flasks containing

10 or 1 mM DFMO as in control

their normal inhibitors.

growth

described

indicating

to the effects

loss of greater

knowledge,

proliferation

et al (18) for a variant by the selective HMO,,

pressure

depletion.

with

HMO,

cells.

in spite of significant

of ODC to the

The data reported

by

Pu and Sd content,

Their observation polyamine

depletion

of normal is, to our

The cell line used in that study, however, pressure

at

QMO or DFMO does result in

than 90% of control

by selective

of

can proliferate

clone as “resistant

of polyamine of HMO,

in

we feel it may be more appropriately

do act on HMO,

the first such report.

clone produced

or more than

even in the presence

refer to the HMO,

that ODC inhibitors

rates of cellular

by Mamont,

These cells, designated

show that incubation

almost immediate

in spite of the

flasks at 96 hours post treatment.

rate for several generations

as resistant

with 10 or

of polyamines,

cell line produced

effect of ODC inhibitors,”

these authors

variant

“MO.

While these authors

anti-proliferative

reported

tissue culture with

Sp

of Sd plus Sp, there were as many cells present

results were recently

incubation

cells treated In addition,

complement

half their normal

clone of the hepatoma

of 9L cells.

and the absence of increased Nonetheless,

loss of more than 2/3 of the cells’ normal

continual

our previous

and total Sd plus Sp content

from DFMO treatment.

1 mM DFMO proliferate

Similar

argue against

5 and 6 show that both 10 and 1 mM DFMO significantly

This is due to both the greater content

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

rather

than a wild-type

was a

cell line such

as 9L. As we have discussed previously to study the role(s) of polyamines the intracellular intracellular

Pu content Sd levels.

not play a crucial unperturbed needed

our observations

in cell proliferation

among cell lines used

and division.

For example,

of unperturbed

9L cells is particularly

The results we report

here may indicate

that polyamines

or may, alternatively,

imply that

role in 9L cell replication

9L cells produce

to support

(17), 9L may be unique

nearly two orders of magnitude

their continued

proliferation.

may also be a characteristic

1197

Either

high relative

to do

more Pu and Sd than

of these explanations

unique to 9L cells.

for

Vol. 86, No. 4, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES 1. Williams-Ashman, H.G., Corti, A., and Tadolini, B. (1976)Ital. J. Biochem., 25, 5-32. 2. Abdel-Monem, M.M., Newton, N.E., and Weeks, C.E. (1974)J. Med. Chem., E, 447-451. 3. Harik, S.I. and Snyder, S.H. (1973)Biochim. Biophys. Acta., 327, 501-509. 4. Abdel-Monem, M.M., Newton, N.E., and Weeks, C.E. (1975)3. Med. Chem., 18, 945-948. 5. Relyea, N. and Rando, R.R. (1975) Biochem. Biophys. Res. Comm., 67, 392-402. 6. Heller, J.S., Canellakis, ES., Bussolotti, D.L., and Coward, J.K., (1975) Biochim. Biophys. Acta, %,197-207. 7. Mamont, P.S., Duchesne, M.C., Grove, J., and Bey, P. (1978). Biochem. Biophys. Res. Comm., 81, 58-66. 8. Bey, P., and Vevert, J.P. (1978)Tet. Letters, 1215-1218. 9. Metcalf, B.W., Bey, P., Danzin, C., Jung, M.J., Casara, P. and Vevert, J.P. (1978)J. Am. Chem. Sot., 100, 2551-2553. 10. Bey, P., Danzin, C., VanDorsselau, V., Mamont, P., Jung, M. and Tardif, C. (1978)J. Med. Chem., 2l, 50-55. II. Prakash, N.J., Schechter, P.J., Grove, J. and Koch-Weser, J. (1978) Cancer Res., 38, 3059-3062. 12. Cohen, S.S. (1971)Introduction to the Polyamines, Prentice-Hall, Englewood Cliffs, New Jersey. 13. Bachrach, U. (1973)Function of Naturally Occurring Polyamines, Academic Press,New York. 14. Heby., O., Marton, L.J., Wilson, C.B., and Martinez, H.M. (1975) J. Cell. Physiol, 86, 511-522. 15. Heby., O., Marton, L.J., Wilson, C.B., and Martinez, H.M. (1975) FEBS Letters, 50, l-4. 16. Marton, L.J. and Lee, P.Y.L. (1975)Clin. Chem., z, 1721-1724. 17. Seidenfeld, J. and Marton, L-J., submitted. IX. Mamont, P.S. Duchesne,M.C., Grove, J., and Tardif, C. (1978)Exp. Cell Res., ll5, 387-393.

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